Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 7, 2015; 21(1): 164-176
Published online Jan 7, 2015. doi: 10.3748/wjg.v21.i1.164
Establishment and characterization of cell lines from chromosomal instable colorectal cancer
Claudia Maletzki, Michael Gock, Martin Randow, Ernst Klar, Maja Huehns, Friedrich Prall, Michael Linnebacher
Claudia Maletzki, Martin Randow, Michael Linnebacher, Section of Molecular Oncology and Immunotherapy, University of Rostock, D-18057 Rostock, Germany
Michael Gock, Ernst Klar, Department of General Surgery, University of Rostock, D-18057 Rostock, Germany
Maja Huehns, Friedrich Prall, Institute of Pathology, University of Rostock, D-18057 Rostock, Germany
Author contributions: Klar E critically revised the paper; Maletzki C and Gock M contributed equally to this work; Maletzki C and Linnebacher M designed the research; Maletzki C and Randow M performed the research; Huehns M and Prall F performed molecular pathology and histology; Maletzki C, Gock M, Randow M and Linnebacher M analyzed the data; and Maletzki C, Gock M and Linnebacher M wrote the paper.
Supported by German Cancer Aid, No. 108919
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Michael Linnebacher, PhD, Section of Molecular Oncology and Immunotherapy, University of Rostock, Schillingallee 35, D-18057 Rostock, Germany. michael.linnebacher@med.uni-rostock.de
Telephone: +49-381-4946013 Fax: +49-381-4946002
Received: May 22, 2014
Peer-review started: May 23, 2014
First decision: June 18, 2014
Revised: June 26, 2014
Accepted: July 24, 2014
Article in press: July 26, 2014
Published online: January 7, 2015
Processing time: 229 Days and 16.7 Hours
Abstract

AIM: To generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions.

METHODS: Cell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure. A comprehensive characterization was subsequently performed. This included quality control, consisting of the confirmation of human and colorectal cancer (CRC) origin by DNA fingerprint and epithelial cell adhesion molecule (EpCAM) staining, as well as mycoplasma and human virus testing. Phenotypic analysis was done by light microscopy and multicolor flow cytometry. Histopathological examination (β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues. Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations, assessment of chromosomal and microsatellite instability, and the grade of CpG island methylation. Additionally, an array-based comparative genomic hybridization analysis was performed. Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers. Finally, tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.

RESULTS: Herein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC. One cell line was derived directly from an early stage case (HROC18), whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor (HROC32). All cell lines were free of contaminating mycoplasma and viruses. Molecular-pathological analysis allowed all cell lines to be classified as chromosomal instable (CIN+). They were aneuploid, with CpG island promoter methylation and microsatellite instability being absent. The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APCmut, p53mut, K-raswt; HROC32: APCwt, p53mut, K-rasmut. All cell lines were characterized as epithelial (EpCAM+) cells, showing distinct morphology and migration speed, but comparable growth kinetics. The cell lines showed different patterns of response towards clinically approved and novel drugs, with HROC18 being more resistant than HROC32 cells. Finally, in vivo tumorigenicity was demonstrated.

CONCLUSION: We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN+ phenotype.

Keywords: Patient-derived tumor model; Colorectal cancer; Individualized medicine; Molecular characterization; Drug response; Translational research

Core tip: We ultra-low passage and well-characterized tumor models are considered the basis for modern preclinical research, but are still rare for colorectal carcinoma (CRC). Herein describe two novel ultra-low passage patient-derived CRC cell lines, HROC18 and HROC32, which were established both direct from patient material and after xenografting. We characterized these models according to phenotype, molecular, morphological, and growth characteristics, as well as by drug response profiles. These cell lines expand our comprehensive collection of tumor models, which in summary provide a useful instrument for basic and translational research. The models are available on request.